Adenosine is known to modulate a number of physiological functions. At the cardiovascular system level, adenosine is strong vasodilator and cardiac depressor. In the central nervous system, adenosine induces sedative, anxiolytic and antiepileptic effects. At the kidney level, it exerts a diphasic action, inducing vasoconstriction at low concentrations and vasodilation at high doses. Adenosine acts as a lipolysis inhibitor on fat cells and as an anti-aggregant on platelets (Stone T. W., Purine Receptors and their Pharmacological Roles, Advances in Drug Research, Academic Press Limited, 1989, 18, 291-429; Progress Cardiovasc. Dis. 1989, 32, 73-97).
A number of studies have shown that adenosine actions are mediated by two subtypes of receptors that are located on the cell membrane: one of high-affinity, inhibiting the activity of the enzyme adenylate cyclase (A1 receptor), and another of low-affinity, stimulating the activity of the same enzyme (A2 receptor). (J. Med. Chem. 1982, 25, 197-207; Physiol. Rev. 1990, 70(3), 761-845; J. Med. Chem. 1992, 35, 407-422). Both receptors are widely spread in the different systems of the organism. In some tissues, however, only one of said receptors is mainly present. For example, the A1 receptor is more prevalent than the A2 receptor at the cardiac level, whereas the A2 receptor is more prevalent than the A1 receptor at the vascular level and on platelets.
Compounds capable of interacting selectively with either the A1 or A2 receptor could have an interesting pharmacological pattern. Furthermore, the vasodilating activity, together with the anti-aggregating action, of the compounds that interact with the A2 receptors may lead to useful therapeutic applications in the treatment of severe cardiovascular pathologies, such as ischemic cardiopathy, hypertension and atherosclerosis. Moreover, due to the actions on central nervous system, the use of A2 selective medicaments can be envisaged in the treatment of cerebrovascular ischemia, epilepsy and various emotional disorders, such as anxiety and psychosis.
Previously, adenosine-5′-N-ethyluronamide or NECA (Mol. Pharmacol., 1986, 25, 331-336) was the only known compound, other than adenosine, having agonist activity at the A2 receptor. Unfortunately, NECA is also active on the A1 receptor and thus lacks specificity for the A2 receptors alone. Because it was the only available compound having A2 affinity, NECA was used for pharmacological tests for the receptor binding.
More recently, however, certain NECA derivatives having A2 receptor selectivity have been developed. These compounds are NECA derivatives that are substituted at the C2-position with phenylamino groups. For example, the compound 2-(p-(carboxyethyl)phenylethylamino)-5′-N-ethyluronamide, named CGS 21680 (J. Pharmacol Exp. Ther., 1989, 251, 888-893), has become the reference compound for the pharmacological studies on A2 receptor.
Other purine derivatives having selective A2 agonist activity are disclosed, for example, in GB-A-2203149; EP-A-0309112; EP-A-0267878; EP-A-0277917; and EP-A-0323807. Substitution at the 2-position of the purine group has been considered promising to give the desired selectivity (J. Med. Chem. 1992, 35, 407-422). 2-Alkynylpurine derivatives have been disclosed in EP-A-0219876 and U.S. Pat. No. 4,956,345.
U.S. Pat. No. 5,593,975 discloses 2-alkynyl adenosine derivatives substituted at the ethyne position with aryl, heterocyclic or hydroxyalkyl groups and in which the riboside residue is substituted by the N-alkyl- (or cycloalkyl)-uronamido. It is reported that these compounds exhibit strong A2 agonist selectivity and, therefore, are useful for the treatment of cardiovascular pathologies, such as cardiac ischemia, hypertension and atherosclerosis and of diseases of the central nervous system, such as cerebrovascular ischemia, epilepsy and emotional disorders (anxiety and psychosis).
The 2-alkynyl adenosine derivatives of U.S. Pat. No. 5,593,975 have the following general formula:

wherein R is hydrogen, C1-C6 alkyl, C3-C7 cycloalkyl, phenyl C1-C3 alkyl;
wherein R1 has one of the following meanings:    (a) phenyl or naphthyl optionally substituted with one to three halogen atoms (chlorine, fluorine or bromine), C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, C2-C6 alkoxycarbonyl, C2-C6 alkoxyalkyl, C1-C6 alkylthio, thio, CHO, cyanomethyl, nitro, cyano, hydroxy, carboxy, C2-C6 acyl, amino, C1-C3 monoalkylamino, C2-C6 dialkylamino, methylenedioxy; or aminocarbonyl;    (b) a group of the formula —(CH2)m-Het wherein m is 0 or an integer from 1 to 3 and Het is 5 or 6 membered heterocyclic aromatic or non aromatic ring, optionally benzocondensed, containing 1 to 3 heteroatoms selected from oxygen, nitrogen or sulphur, linked through a carbon atom or through a nitrogen atom;    (c) C3-C7 cycloalkyl optionally containing unsaturations or C2-C4 alkenyl;    (d) moieties of the following formula:
                where R2 is hydrogen, methyl or phenyl;                    R4 is OH, NH2, dialkylamino, halogen, or cyano;            R5 is hydrogen, C1-C6 linear or branched alkyl, C5-C6 cycloalkyl or C3-C7 cycloalkenyl, phenyl-C1-C2-alkyl            or R2 and R5, taken together, form a 5 or 6-membered carbocyclic ring or R3 is hydrogen and R2 and R4, taken together, form an oxo group or a corresponding acetalic derivative;            when R is different from hydrogen and/or R3 is different from ethyl, R1 can also be C1-C6 linear or branched alkyl; and            n is 0 or 1 to 4; and                        wherein R3 is C1-C6 alkyl, C3-C7-cycloalkyl, phenyl or benzyl; provided that when R is different that hydrogen or when R is hydrogen and R3 is cyclopentyl, phenyl or benzyl, R1 can also be C1-C6 linear or branched alkyl.        
The 2-alkynyl adenosine compounds of U.S. Pat. No. 5,593,975 are prepared by the general synthetic schemes shown below.


In schemes I and II, R′ and R′1 have the same meanings as R and R1, respectively, or they are groups which can be converted into R and R1, respectively, for example, by removing any protecting groups which can be present in R′ and R′1 compatible with the reaction conditions; Y is Br or I and X is chlorine, bromine or iodine.
The reactions shown in schemes I and II are carried out in the presence of catalysts (for example: bis(triphenylphosphine) palladium dichloride and a cuprous halide) and a suitable acid-binding agent, such as an organic base (for example: triethylamine, diisopropylethylamine or pyridine).
As the solvent, a substituted amide (such as dimethylformamide), an ether (such as dioxane or tetrahydrofuran), acetonitrile or optionally a mixture of two or more of said solvents, are preferably used.
The compounds of formula II, in which Y is iodine and R′ is hydrogen, can be prepared from 2-iodoadenosine (Nair et al., Synthesis, 1982, 670-672) according to the following Scheme III:

Compounds of formula VIII in which Y is iodine and R′ is different from hydrogen can be prepared according to the following Scheme IV:

In the above Scheme IV, R, R1 and R3 are as above defined.
The compounds of formula V are prepared by reaction of compounds of formula II with an acetylene derivative, for example, 1-trimethylsilylacetylene, under the conditions reported for the reaction between compounds II and III. Compounds III and IV are known or they can be prepared according to well-known methods.
U.S. Pat. No. 6,322,771 discloses compounds of formula IX:

wherein                (a) each R is individually hydrogen, C1-C6 alkyl, C3-C7 cycloalkyl, phenyl or phenyl(C1-C3)-alkyl;        (b) X is —CH2OH, —CO2R2, —OC(O)R2, —CH2OC(O)R2 or —C(O)NR3R4;        (c) each of R2, R3 and R4 is individually H, C1-C6-alkyl; C1-C6-alkyl substituted with 1-3 C1-C6 alkoxy, C3-7cycloalkyl, C1-C6-alkylthio, halogen, hydroxy, amino, mono(C1-C6-alkyl)amino, di(C1-C6-alkyl)amino, or C6-C10-aryl, wherein aryl may be substituted with 1-3 halogen, C1-C6-alkyl, hydroxy, amino, mono(C1-C6-alkyl)amino, or di(C1-C6-alkyl)amino; C6-C10-aryl; or C6-C10-aryl substituted with 1-3 halogen, hydroxy, amino, mono(C1-C6-alkyl)amino, di(C1-C6-alkyl)amino, or C1-C6-alkyl; and        (d) R1 is (X—(Z)—)n[(C3-C10)cycloalkyl]-(Z′)— wherein Z and Z′ are individually (C1-C10)alkyl, optionally interrupted by 1-3 S or nonperoxide O, or is absent, and n is 1-3.It is disclosed that the compounds of Formula IX may be prepared by the synthetic methods disclosed in U.S. Pat. Nos. 5,278,150; 5,140,015; 5,877,180; 5,593,975; and 4,956,345.        
U.S. Pat. No. 6,322,771 discloses preferred compounds of Formula IX, wherein each R is H, X is ethylaminocarbonyl and R1 is 4-carboxycyclohexylmethyl (DWH-146a), R1 is 4-methoxycarbonylcyclohexylmethyl (DWH-146e) or R1 is 4-acetoxymethyl-cyclohexylmethyl (JMR-193):

According to U.S. Pat. No. 6,322,771, the synthesis of the methyl 4[3-(6-amino-9(5-[(ethylamino)carbonyl]-3,4-dihydroxytetrahydro-Z-furanyl-9H-2-purinyl)-2-propynyl]-1-cyclohexanecarboxylate (DWH-146e) was accomplished by the cross coupling of an iodo-adenosine derivative (N-ethyl-1′-deoxy-1′-(amino-2-iodo-9H-purin-9-yl)-β-D-ribofuranuoramide) with methyl 4-(2-propynyl)-1-cyclohexanecarboxylate by utilization of a Pd(II) catalyst.
The iodo-adenosine derivative was first prepared from guanosine by treating it with acetic anhydride, which acetylates the sugar hydroxyls. The resulting compound was then chlorinated at position 6 with tetramethyl ammonium chloride and phosphorous oxychloride. Iodination of position 2 was accomplished via a modified Sandmeyer reaction, followed by displacement of the 6-Cl and sugar acetates with ammonia. The 2′ and 3′ hydroxyls were protected as the acetonide and the 5′ hydroxyl was oxidized to the acid with potassium permanganate. Deprotection of the 2′ and 3′ acetonide, Fisher esterification of the 5′ acid with ethanol and conversion of the resulting ethyl ester to the ethyl amide with ethylamine gave N-ethyl-1′-deoxy-1′-(amino-2-iodo-9H-purin-9-yl)-β-D-ribofuranuoramide.
The acetylene [methyl 4-(2-propynyl)-1-cyclohexanecarboxylate] was synthesized starting from trans-1,4-cyclohexanedimethanol. Initially, the trans-diol was monotosylated followed by displacement of the tosylate with an acetylene anion. The hydroxyl of the resulting hydroxyl acetylene species was oxidized to the acid via Jones reagent followed by methylation with (trimethylsilyl)diazomethane to give methyl 4-(2-propynyl)-1-cyclohexanecarboxylate.
A cross-coupling reaction of N-ethyl-1′-deoxy-1′-(amino-2-iodo-9H-purin-9-yl)-β-D-ribofuranuoramide and methyl 4-(2-propynyl)-1-cyclohexanecarboxylate was then performed. To a solution of N,N-dimethylformamide (0.5 mL), acetonitrile (1 mL), triethylamine (0.25 mL), and N-ethyl-1′-deoxy-1′-(amino-2-iodo-9H-purin-9-yl)-β-D-ribofuranuroamide (25 mg, 0.06 mmol) was added bis(triphenylphosphine)palladium dichloride (1 mg, 2 mol %) and copper(I) iodide (0.06 mg, 0.5 mol %). To the resulting mixture was added methyl 4-(2-propynyl)-1-cyclohexanecarboxylate (54 mg, 0.3 mmol) and the reaction was stirred under nitrogen atmosphere for 16 hours. The solvent was removed under vacuum and the resulting residue was flash chromatographed in 20% methanol in chloroform (Rf=0.45) to give 19 mg (off-white solid, mp 125° C. (decomposed)) of 4[3-(6-amino-9(5-[(ethylamino)carbonyl]-3,4-dihydroxytetrahydro-Z-furanyl)-9H-2-purinyl)-2-propynyl]-1-cyclohexanecarboxylate (DWH-146e).
These above-described synthetic methods for producing 2-alkynyladenosine derivatives, including DWH-146e, provide lower yields than desired, require prolonged reaction times and require extensive chromatographic purification. For example in U.S. Pat. No. 5,593,975, the acetonide protection (first step of Scheme III above) requires purification by column chromatography. Furthermore, the oxidation procedure (second step of Scheme III above) requires prolonged reaction times and has been noted to be troublesome due to competing oxidation at the 2-iodo position. Homma et al., J. Med. Chem., 1992, 35, 2881-2890. In U.S. Pat. No. 6,322,771, the first step of the synthesis of the cyclohexane-containing acetylene requires purification by column chromatography to separate the desired mono-tosyl product from the starting diol and bis-tosyl products. In the second step of the synthesis of the cyclohexane-containing acetylene, prolonged reaction times, a large excess of the acetylene anion and purification by column chromatography are required. Furthermore, the cross-coupling reaction between the cyclohexane-containing acetylene and 2-iodoNECA proceeds with poor yield after chromatography.
Thus, there is a need for improved synthetic methods for producing 2-alkynyladenosine derivatives that provide higher yields and require less purification than prior art methods. Using alternative reagents and steps, we have discovered an improved method for producing 2-alkynyladenosine derivatives at higher yields with less chromatographic purification required.